Disease Resistant Plants

ABSTRACT

The present invention relates to a plant, which is resistant to a pathogen of viral, bacterial, fungal or oomycete origin, wherein the plant has an increased homoserine level as compared to a plant that is not resistant to the said pathogen, in particular organisms of the phylum Oomycota. The invention further relates to a method for obtaining a plant, which is resistant to a pathogen of viral, bacterial, fungal or oomycete origin, comprising increasing the endogenous homoserine level in the plant.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional application of copending U.S. patentapplication Ser. No. 13/545,853, filed Jul. 10, 2012, which is adivisional application of U.S. patent application Ser. No. 12/092,253,filed Dec. 19, 2008, and issued as U.S. Pat. No. 8,237,019, which is aU.S. National Phase application filed under 35 U.S.C. §371 claimingpriority to PCT Application No. PCT/EP2006/010535, filed Nov. 1, 2006and which claims priority to PCT Application No. PCT/EP2005/011718,filed Nov. 1, 2005, each of which is incorporated herein in reference intheir entirety.

The Sequence Listing associated with this application is filed inelectronic format via EFS-Web and is hereby incorporated by referenceinto the specification in its entirety. The name of the text filecontaining the Sequence Listing is 123498_ST25.txt. The size of the textfile is 90,740 bytes, and the text file was created on Dec. 5, 2012.

BACKGROUND OF THE INVENTION

The present invention relates to disease resistant plants, in particularplants resistant to organisms of the phylum Oomycota, the oomycetes. Theinvention further relates to plant genes conferring disease resistanceand methods of obtaining such disease resistant plants for providingprotection to Oomycota pathogens.

Resistance of plants to pathogens has been extensively studied, for bothpathogen specific and broad resistance. In many cases resistance isspecified by dominant genes for resistance. Many of these race-specificor gene-for-gene resistance genes have been identified that mediatepathogen recognition by directly or indirectly interacting withavirulence gene products or other molecules from the pathogen. Thisrecognition leads to the activation of a wide range of plant defenseresponses that arrest pathogen growth.

In plant breeding there is a constant struggle to identify new sourcesof mostly monogenic dominant resistance genes. In cultivars with newlyintroduced single resistance genes, protection from disease is oftenrapidly broken, because pathogens evolve and adapt at a high frequencyand regain the ability to successfully infect the host plant. Therefore,the availability of new sources of disease resistance is highly needed.

Alternative resistance mechanisms act for example through the modulationof the defense response in plants, such as the resistance mediated bythe recessive mlo gene in barley to the powdery mildew pathogen Blumeriagraminis f. sp. hordei. Plants carrying mutated alleles of the wildtypeMLO gene exhibit almost complete resistance coinciding with the abortionof attempted fungal penetration of the cell wall of single attackedepidermal cells. The wild type MLO gene thus acts as a negativeregulator of the pathogen response. This is described in WO9804586.

Other examples are the recessive powdery mildew resistance genes, foundin a screen for loss of susceptibility to Erysiphe cichoracearum. Threegenes have been cloned so far, named PMR6, which encodes a pectatelyase-like protein, PMR4 which encodes a callose synthase, and PMR5which encodes a protein of unknown function. Both mlo and pmr genesappear to specifically confer resistance to powdery mildew and not tooomycetes such as downy mildews.

Broad pathogen resistance, or systemic forms of resistance such as SAR,has been obtained by two main ways. The first is by mutation of negativeregulators of plant defense and cell death, such as in the cpr, lsd andacd mutants of Arabidopsis. The second is by transgenic overexpressionof inducers or regulators of plant defense, such as in NPR1overexpressing plants.

The disadvantage of these known resistance mechanisms is that, besidespathogen resistance, these plants often show detectable additional andundesirable phenotypes, such as stunted growth or the spontaneousformation of cell death.

It is an object of the present invention to provide a form of resistancethat is broad, durable and not associated with undesirable phenotypes.

In the research that led to the present invention, an Arabidopsisthaliana mutant screen was performed for reduced susceptibility to thedowny mildew pathogen Hyaloperonospora parasitica. EMS-mutants weregenerated in the highly susceptible Arabidopsis line Ler eds1-2. Eightdowny mildew resistant (dmr) mutants were analysed in detail,corresponding to 6 different loci. Microscopic analysis showed that inall mutants H. parasitica growth was severely reduced. Resistance ofdmr3, dmr4 and dmr5 was associated with constitutive activation of plantdefence. Furthermore, dmr3 and dmr4, but not dmr5, were also resistantto Pseudomonas syringae and Golovinomyces orontii.

In contrast, enhanced activation of plant defense was not observed inthe dmr1, dmr2, and dmr6 mutants. The results of this research have beendescribed in Van Damme et al. (2005) Molecular Plant-MicrobeInteractions 18(6) 583-592. This article does however not disclose theidentification and characterization of the DMR genes.

BRIEF SUMMARY OF THE INVENTION

According to the present invention it was now found that DMR1 is thegene encoding homoserine kinase (HSK). For Arabidopsis five differentmutant dmr1 alleles have been sequenced each leading to a differentamino acid change in the HSK protein. HSK is a key enzyme in thebiosynthesis of the amino acids methionine, threonine and isoleucine andis therefore believed to be essential. The various dmr1 mutants showdefects in HSK causing the plants to accumulate homoserine The fivedifferent alleles show different levels of resistance that correlate todifferent levels of homoserine accumulation in the mutants.

The present invention thus provides a plant, which is resistant to apathogen of viral, bacterial, fungal or oomycete origin, characterizedin that the plant has an altered homoserine level as compared to a plantthat is not resistant to the said pathogen.

This form of resistance is in particular effective against pathogens ofthe phylum Oomycota, such as Albugo, Aphanomyces, Basidiophora, Bremia,Hyaloperonospora, Pachymetra, Paraperonospora, Perofascia,Peronophythora, Peronospora, Peronosclerospora, Phytium, Phytophthora,Plasmopara, Protobremia, Pseudoperonospora, Sclerospora, Viennotiaspecies.

The resistance is based on an altered level of homoserine in planta.More in particular, the resistance is based on an increased level ofhomoserine in planta. Such increased levels can be achieved in variousways.

First, homoserine can be provided by an external source. Second, theendogenous homoserine level can be increased. This can be achieved bylowering the enzymatic activity of the homoserine kinase gene whichleads to a lower conversion of homoserine and thus an accumulationthereof. Alternatively, the expression of the homoserine kinase enzymecan be reduced. This also leads to a lower conversion of homoserine andthus an accumulation thereof. Another way to increase the endogenoushomoserine level is by increasing its biosynthesis via the aspartatepathway. Reducing the expression of the homoserine kinase gene can initself be achieved in various ways, either directly, such as by genesilencing, or indirectly by modifying the regulatory sequences thereofor by stimulating repression of the gene.

Modulating the HSK gene to lower its activity or expression can beachieved at various levels. First, the endogenous gene can be directlymutated. This can be achieved by means of a mutagenic treatment.Alternatively, a modified HSK gene can be brought into the plant bymeans of transgenic techniques or by introgression, or the expression ofHSK can be reduced at the regulatory level, for example by modifying theregulatory sequences or by gene silencing.

In one embodiment of the invention, an increase (accumulation) inhomoserine level in the plant is achieved by administration ofhomoserine to the plant. This is suitably done by treating plants withL-homoserine, e.g. by spraying or infiltrating with a homoserinesolution.

Treatment of a plant with exogenous homoserine is known from WO00/70016.This publication discloses how homoserine is applied to a plantresulting in an increase in the phenol concentration in the plant. Thepublication does not show that plants thus treated are resistant topathogens. In fact, WO00/70016 does not disclose nor suggest that anincrease in endogenous homoserine would lead to pathogen resistance.

Alternatively, endogenous homoserine is increased by modulating plantamino acid biosynthetic or metabolic pathways.

In one embodiment, the increased endogenous production is the result ofa reduced endogenous HSK gene expression thus leading to a lessefficient conversion of homoserine into phospho-homoserine and thesubsequent biosynthesis of methionine and threonine. This reducedexpression of HSK is for example the result of a mutation in the HSKgene leading to reduced mRNA or protein stability.

In another embodiment reduced expression can be achieved bydownregulation of the HSK gene expression either at the transcriptionalor the translational level, e.g. by gene silencing or by mutations inthe regulatory sequences that affect the expression of the HSK gene. Anexample of a method of achieving gene silencing is by means of RNAi.

In a further embodiment the increase in endogenous homoserine level canbe obtained by inducing changes in the biosynthesis or metabolism ofhomoserine. In a particular embodiment this is achieved by mutations inthe HSK coding sequence that result in a HSK protein with a reducedenzymatic activity thus leading to a lower conversion of homoserine intophospho-homoserine. Another embodiment is the upregulation of genes inthe aspartate pathway causing a higher production and thus accumulationof L-homoserine in planta.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows orthologous HSK sequences that have been identified inpublicly available databases and obtained by PCR amplification on cDNAand subsequent sequencing. FIG. 1 shows the alignment of the amino acidsequences of the HSK proteins of Arabidopsis thaliana and orthologs fromCitrus sinensis, Populus trichocarpa (1), Populus trichocapa (2),Solanum tuberosum (2), Vitis vinifera, Lactuca sativa, Solanum tuberosum(1), Solanum lycopersicum, Nicotiana benthamiana, Ipomoea nil, Glycinemax, Phaseolus vulgaris, Cucumis sativus, Spinacia oleracea, Pinustaeda, Zea mays, and Oryza sativa using the CLUSTAL W (1.82) multiplesequence alignment programme (EBI). Below the sequences the conservedamino acids are indicated by the dots, and the identical amino acids areindicated by the asterisks. The black triangles and corresponding textindicate the amino acids that are substituted in the five Arabidopsisdmr mutants.

Table 2 shows the Genbank accession numbers and GenInfo identifiers ofthe Arabidopsis HSK mRNA and orthologous sequences from other plantspecies.

FIG. 2 shows the percentage of conidiophore formation by twoHyaloperonospora parasitica isolates, Cala2 and Waco9, on the mutantsdmr1-1, dmr1-2, dmr1-3 and dmr1-4 and the parental line, Ler eds1-2, 7days post inoculation. The conidiophores formed on the parental linewere set to 100%.

FIG. 3 is a graphic overview of the three major steps in the cloning ofDMR1. a) Initial mapping of dmr1 resulted in positioning of the locus onthe lower arm of chromosome 2 between positions 7.42 and 7.56 Mb. Threeinsert/deletion (INDEL) markers were designed (position of the markersF6P23, T23A1 and F5J6 is indicated by the black lines). These markerswere used to identify recombinants from several 100 segregating F2 andF3 plants. Primer sequences of these INDEL markers and additionalmarkers to identify the breakpoints in the collected recombinants ispresented in table 3. b) One marker, At2g17270 (indicated by the greyline), showed the strongest linkage with resistance. The dmr1 locuscould be further delimited to a region containing 8 genes,at2g17250-at2g17290. The eight genes were amplified and sequenced tolook for mutations in the coding sequences using the primers describedin table 4. DNA sequence analysis of all 8 candidate genes led to thediscovery of point mutations in the At2g17265 gene in all 5 dmr1mutants. c) Each dmr1 mutant has a point mutation at a differentlocation in the At2g17265 gene, which encodes homoserine kinase.

FIG. 4 shows a schematic drawing of the HSK coding sequence and thepositions and nucleotide substitutions of the 5 different dmr1 mutationsin the HSK coding sequence (the nucleotide positions, indicated by theblack triangles, are relative to the ATG start codon which start onposition i). The 5′UTR and 3′UTR are shown by light grey boxes. Belowthe nucleotide sequence the protein sequence is shown. The HSK proteincontains a putative transit sequence for chloroplast targeting (darkgrey part). The amino acid changes resulting from the 5 dmr1 mutationsare indicated at their amino acid (aa) position number (black triangles)in the HSK protein.

FIG. 5 shows the position of the homoserine kinase enzyme in theaspartate pathway for the biosynthesis of the amino acids threonine,methionine and isoleucine.

FIG. 6 shows the number of conidiophores per Ler eds 1-2 seedlings 5days post inoculation with two different isolates of H. parasitica,Waco9 and Cala2. The inoculated seedlings were infiltrated with dH2O,D-homoserine (5 mM) or L-homoserine (5 mM) at 3 days post inoculationwith the pathogen. Seedlings treated with L-homoserine show a completeabsence of conidiophore formation and are thus resistant.

FIG. 7 shows the growth and development of H. parasitica in seedlingstreated with water, D-homoserine (5 mM), or L-homoserine (5 mM) asanalysed by microscopy of trypan blue stained seedlings.

a: Conidiophore formation after HS treatment on Ler ed1-2 seedlings (10×magnification). No conidiophore formation was detected afterL-homoserine infiltration, whereas control plants show abundantsporulation.

b: Haustorial development is affected by L-homoserine (5 mM)infiltration (40× magnification), but not in plants treated with wateror D-homoserine.

FIGS. 8 and 9 show the nucleotide and amino acid sequence of thehomoserine kinase gene (At2g17265, NM_(—)127281, GI:18398362) andprotein (At2g17265, NP_(—)179318, GI: 15227800) of Arabidopsis thaliana,respectively (SEQ ID NOs: 99-100).

FIG. 10 shows the nucleotide and the predicted amino acid sequence ofthe homoserine kinase coding sequence (CDS) and protein, respectively,of Lactuca sativa (SEQ ID NOs. 101-102)

FIG. 11 shows the nucleotide and the predicted amino acid sequence ofthe homoserine kinase coding sequence (CDS) and protein, respectively,of Vitis vinifera (SEQ ID NOs: 103-104)

FIG. 12 shows the nucleotide and the predicted amino acid sequence ofthe homoserine kinase coding sequence (CDS) and protein, respectively,of Cucumis sativus (SEQ ID NOs: 105-106)

FIG. 13 shows the nucleotide and the predicted amino acid sequence ofthe homoserine kinase coding sequence (CDS) and protein, respectively,of Spinacia oleracea (SEQ ID NOs: 107-108)

FIG. 14 shows the nucleotide and the predicted amino acid sequence ofthe homoserine kinase coding sequence (CDS) and protein, respectively,of Solanum lycopersicum (SEQ ID NOs: 109-110)

DETAILED DESCRIPTION

This invention is based on research performed on resistance toHyaloperonospora parasitica in Arabidopsis but is a general concept thatcan be more generally applied in plants, in particular in crop plantsthat are susceptible to infections with pathogens, such as Oomycota.

The invention is suitable for a large number of plant diseases caused byoomycetes such as, but not limited to, Bremia lactucae on lettuce,Peronospora farinosa on spinach, Pseudoperonospora cubensis on membersof the Cucurbitaceae family, e.g. cucumber, Peronospora destructor ononion, Hyaloperonospora parasitica on members of the Brasicaceae family,e.g. cabbage, Plasmopara viticola on grape, Phytophthora infestans ontomato and potato, and Phytophthora sojae on soybean.

The homoserine level in these other plants can be increased with alltechniques described above. However, when the modification of the HSKgene expression in a plant is to be achieved via genetic modification ofthe HSK gene or via the identification of mutations in the HSK gene, andthe gene is not yet known it must first be identified. To generatepathogen-resistant plants, in particular crop plants, via geneticmodification of the HSK gene or via the identification of mutations inthe HSK gene, the orthologous HSK genes must be isolated from theseplant species. Orthologs are defined as the genes or proteins from otherorganisms that have the same function.

Various methods are available for the identification of orthologoussequences in other plants.

A method for the identification of HSK orthologous sequences in a plantspecies, may for example comprise identification of homoserine kinaseESTs of the plant species in a database; designing primers foramplification of the complete homoserine kinase transcript or cDNA;performing amplification experiments with the primers to obtain thecorresponding complete transcript or cDNA; and determining thenucleotide sequence of the transcript or cDNA.

Suitable methods for amplifying the complete transcript or cDNA insituations where only part of the coding sequence is known are theadvanced PCR techniques 5′RACE, 3′RACE, TAIL-PCR, RLM-RACE andvectorette PCR.

Alternatively, if no nucleotide sequences are available for the plantspecies of interest, primers are designed on the HSK gene of a plantspecies closely related to the plant of interest, based on conserveddomains as determined by multiple nucleotide sequence alignment, andused to PCR amplify the orthologous sequence. Such primers are suitablydegenerate primers.

Another reliable method to assess a given sequence as being a HSKortholog is by identification of the reciprocal best hit. A candidateorthologous HSK sequence of a given plant species is identified as thebest hit from DNA databases when searching with the Arabidopsis HSKprotein or DNA sequence, or that of another plant species, using a Blastprogramme. The obtained candidate orthologous nucleotide sequence of thegiven plant species is used to search for homology to all Arabidopsisproteins present in the DNA databases (e.g. at NCBI or TAIR) using theBlastX search method. If the best hit and score is to the ArabidopsisHSK protein, the given DNA sequence can be described as being anortholog, or orthologous sequence.

HSK is encoded by a single gene in Arabidopsis and rice as deduced fromthe complete genome sequences that are publicly available for theseplant species. In most other plant species tested so far, HSK appears tobe encoded by a single gene, as determined by the analysis of mRNAsequences and EST data from public DNA databases, except for potato,tobacco and poplar for which two HSK homologs have been identified. Theorthologous genes and proteins are identified in these plants bynucleotide and amino acid comparisons with the information that ispresent in public databases.

Alternatively, if no DNA sequences are available for the desired plantspecies, orthologous sequences are isolated by heterologoushybridization using DNA probes of the HSK gene of Arabidopsis or anotherplant or by PCR methods, making use of conserved domains in the HSKcoding sequence to define the primers. For many crop species, partialHSK mRNA sequences are available that can be used to design primers tosubsequently PCR amplify the complete mRNA or genomic sequences for DNAsequence analysis.

In a specific embodiment the ortholog is a gene of which the encodedprotein shows at least 50% identity with the Arabidopsis HSK protein orthat of other plant HSK proteins. In a more specific embodiment thehomology is at least 55%, more specifically at least 60%, even morespecifically at least 65%, at least 70%, at least 75%, at least 80%, atleast 85%, at least 90%, at least 95% or at least 99%.

After orthologous HSK sequences are identified, the complete nucleotidesequence of the regulatory and coding sequence of the gene is identifiedby standard molecular biological techniques. For this, genomic librariesof the plant species are screened by DNA hybridization or PCR withprobes or primers derived from a known homoserine kinase gene, such asthe above described probes and primers, to identify the genomic clonescontaining the HSK gene. Alternatively, advanced PCR methods, such asRNA Ligase Mediated RACE (RLM-RACE), can be used to directly amplifygene and cDNA sequences from genomic DNA or reverse-transcribed mRNA.DNA sequencing subsequently results in the characterization of thecomplete gene or coding sequence.

Once the DNA sequence of the gene is known this information is used toprepare the means to modulate the expression of the homoserine kinasegene in anyone of the ways described above.

More in particular, to achieve a reduced HSK activity the expression ofthe HSK gene can be down-regulated or the enzymatic activity of the HSKprotein can be reduced by amino acid substitutions resulting fromnucleotide changes in the HSK coding sequence.

In a particular embodiment of the invention, downregulation of HSK geneexpression is achieved by gene-silencing using RNAi. For this,transgenic plants are generated expressing a HSK anti-sense construct,an optimized micro-RNA construct, an inverted repeat construct, or acombined sense-anti-sense construct, so as to generate dsRNAcorresponding to HSK that leads to gene silencing.

In an alternative embodiment, one or more regulators of the HSK gene aredownregulated (in case of transcriptional activators) by RNAi.

In another embodiment regulators are upregulated (in case of repressorproteins) by transgenic overexpression. Overexpression is achieved in aparticular embodiment by expressing repressor proteins of the HSK genefrom a strong promoter, e.g. the 35S promoter that is commonly used inplant biotechnology.

The downregulation of the HSK gene can also be achieved by mutagenesisof the regulatory elements in the promoter, terminator region, orpotential introns. Mutations in the HSK coding sequence in many caseslead to amino acid substitutions or premature stop codons thatnegatively affect the expression or activity of the encoded HSK enzyme.

These and other mutations that affect expression of HSK are induced inplants by using mutagenic chemicals such as ethyl methane sulfonate(EMS), by irradiation of plant material with gamma rays or fastneutrons, or by other means. The resulting nucleotide changes arerandom, but in a large collection of mutagenized plants the mutations inthe HSK gene can be readily identified by using the TILLING (TargetingInduced Local Lesions IN Genomes) method (McCallum et al. (2000)Targeted screening for induced mutations. Nat. Biotechnol. 18, 455-457,and Henikoff et al. (2004) TILLING. Traditional mutagenesis meetsfunctional genomics. Plant Physiol. 135, 630-636). The principle of thismethod is based on the PCR amplification of the gene of interest fromgenomic DNA of a large collection of mutagenized plants in the M2generation. By DNA sequencing or by looking for point mutations using asingle-strand specific nuclease, such as the CEL-I nuclease (Till et al.(2004) Mismatch cleavage by single-strand specific nucleases. NucleicAcids Res. 32, 2632-2641) the individual plants that have a mutation inthe gene of interest are identified.

By screening many plants, a large collection of mutant alleles isobtained, each giving a different effect on gene expression or enzymeactivity. The gene expression or enzyme activity can be tested byanalysis of HSK transcript levels (e.g. by RT-PCR), quantification ofHSK protein levels with antibodies or by amino acid analysis, measuringhomoserine accumulation as a result of reduced HSK activity. Thesemethods are known to the person skilled in the art.

The skilled person can use the usual pathogen tests to see if thehomoserine accumulation is sufficient to induce pathogen resistance.

Plants with the desired reduced HSK activity or expression are thenback-crossed or crossed to other breeding lines to transfer only thedesired new allele into the background of the crop wanted.

The invention further relates to mutated HSK genes encoding HSK proteinswith a reduced enzymatic activity. In a particular embodiment, theinvention relates to the dmr1 alleles dmr1-1, dmr1-2, dmr1-3, dmr1-4 anddmr1-5.

In another embodiment, the invention relates to mutated versions of theHSK genes of Lactuca sativa, Vitis vinifera, Cucumis sativus, Spinaciaoleracea and Solanum lycopersicum as shown in FIGS. 10-14 (SEQ ID NOs:101-110).

The present invention demonstrates that plants having an increasedhomoserine level show resistance to pathogens, in particular of oomyceteorigin. With this knowledge the skilled person can actively modify theHSK gene by means of mutagenesis or transgenic approaches, but alsoidentify so far unknown natural variants in a given plant species thataccumulate homoserine or that have variants of the HSK gene that lead toan increase in homoserine, and to use these natural variants accordingto the invention.

In the present application the terms “homoserine kinase” and “HSK” areused interchangeably.

The present invention is illustrated in the following examples that arenot intended to limit the invention in any way. In the examplesreference is made to the following figures.

EXAMPLES Example 1 Characterization of the Gene Responsible for PathogenResistance in dmr Mutants

Van Damme et al., 2005, supra disclose four mutants, dmr1-1, dmr1-2,dmr1-3 and dmr1-4 that are resistant to H. parasitica. The level ofresistance can be examined by counting conidiophores per seedling leafseven day post inoculation with the H. parasitica Cala2 isolate(obtainable from Dr. E. Holub (Warwick HRI, Wellesbourne, UK or Dr. G.Van den Ackerveken, Department of Biology, University of Utrecht,Utrecht, NL). For the parental line, Ler eds1-2 (Parker et al., 1996.Plant Cell 8:2033-2046), which is highly susceptible, the number ofconidiophores is set at 100%. The reduction in conidiophore formation onthe infected dmr1 mutants compared to seedlings of the parental line isshown in FIG. 2.

According to the invention, the gene responsible for resistance to H.parasitaca in the dmr1 mutants of van Damme el al., 2005, supra has beencloned by a combination of mapping and sequencing of candidate genes.

DMR1 was isolated by map-based cloning. The dmr1 mutants were crossed tothe FN2 Col-0 mutant to generate a mapping population. The FN2 mutant issusceptible to the H. parasitica isolate Cala2, due to a fast neutronmutation in the RPP7A gene (Sinapidou et al., 2004, Plant J.38:898-909). All 5 dmr1 mutants carry single recessive mutations as theF1 plants were susceptible, and approximately a quarter of the F2 plantsdisplayed H. parasitica resistance.

The DMR1 cloning procedure is illustrated in FIG. 3 and described inmore detail below. The map location of the dmr1 locus was firstdetermined by genotyping 48 resistant F2 plants to be located on thelower arm of chromosome 2. From an additional screen for newrecombinants on 650 F2 plants ˜90 F2 recombinant plants between twoINDEL (insertion/deletion) markers on BAC T24112 at 7.2 Mb and BAC F5J6at 7.56 Mb (according to the TIGR Arabidopsis genome release Version 5.0of January 2004) were identified, which allowed to map the gene to aregion containing a contig of 5 BACs.

The F2 plants were genotyped and the F3 generation was phenotyped inorder to fine map the dmr1 locus. The dmr1 mutation could be mapped to a˜130 kb region (encompassing 3 overlapping BAC clones: F6P23, T23A1, andF5J6) between two INDEL markers located on BAC F6P23, at 7.42 Mb andF5J6 at 7.56 Mb (according to the TIGR Arabidopsis genome releaseVersion 5.0 of January 2004). This resulted in an area of 30 putativegene candidates for the dmr1 locus, between the Arabidopsis genes withthe TAIR codes AT2g17060 and AT2g17380. Additionally cleaved amplifiedpolymorphic sequences (CAPS) markers were designed based on SNPs linkedto genes AT2g17190, AT2g17200, AT2g17270, At2g17300, At2g17310 andAt2g17360 genes.

Analyses of 5 remaining recombinants in this region with these CAPSmarker data left 8 candidate genes, At2g17230 (NM_(—)127277,GI:30679913), At2g17240 (NM_(—)127278, GI:30679916), At2g17250(NM_(—)127279, GI:22325730), At2g17260 (NM_(—)127280, GI:30679922).At2g17265 (NM_(—)127281, GI:18398362), At2g17270 (NM_(—)127282,GI:30679927), At2g17280 (NM_(—)127283, GI:42569096), At2g17290(NM_(—)127284, GI:30679934). Sequencing of all the 8 genes resulted inthe finding of point mutations in the AT2g17265 coding gene in the fivedmr1 alleles: dmr1-1, dmr1-2, dmr1-3, dmr1-4 and dmr1-5, clearlydemonstrating that AT2g17265 is DMR1. FIG. 3 shows a scheme of dnrl withpoint mutations of different alleles.

At2g17265 encodes the homoserine kinase (HSK) enzyme, so far the onlyArabidopsis gene exhibiting this function.

In Arabidopsis, HSK is encoded by a single gene. At2g17265 (Lee &Leustek, 1999, Arch. Biochem. Biophys. 372: 135-142). HSK is the fourthenzyme in the aspartate pathway required for the biosynthesis of theamino acids methionine, threonine and isoleucine. HSK catalyzes thephosphorylation of homoserine to homoserine phosphate (FIG. 5).

Example 2 Amino Acid Analysis

Homoserine phosphate is an intermediate in the production of methionine,isoleucine and threonine in Arabidopsis. Since homoserine kinase has akey role in the production of amino acids, free amino acid levels weredetermined in the parental line Ler eds1-2 and the four different dmr1mutants. For this amino acids from total leaves were extracted with 80%methanol, followed by a second extraction with 20% methanol. Thecombined extracts were dried and dissolved in water. After addition ofthe internal standard, S-amino-ethyl-cysteine (SAEC) amino acids weredetected by automated ion-exchange chromatography with post columnninhydrin derivatization on a JOEL AminoTac JLC-500/V (Tokyo, Japan).

Amino acid analysis of four different dmr1 mutants and the parentalline, Ler eds 1-2 showed an accumulation of homoserine in the dmr1mutants, whereas this intermediate amino acid was not detectable in theparental line Ler eds1-2. There was no reduction in the level ofmethionine, isoleucine and threonine in the dmr1 mutants (Table 1).

TABLE 1 Concentration (in pmol/mg fresh weight) of homoserine,methionine, threonine and isoleucine in above-ground parts of 2-week oldseedlings of the parental line Ler eds 1-2 and the mutants dmr1-1,dmr1-2, dmr1-3 and dmr1-4. Homoserine Methionine Isoleucine Threoninedmr1-1 964 29 12 264 dmr1-2 7128 14 29 368 dmr1-3 466 11 16 212 dmr1-46597 11 32 597 Ler eds 1-2 0 7 10 185Due to the reduced activity of the HSK in the dmr1 mutants, homoserineaccumulates. This effect could be further enhanced by a stronger influxof aspartate into the pathway leading to an even higher level ofhomoserine. The high concentration of the substrate homoserine wouldstill allow sufficient phosphorylation by the mutated HSK so that thelevels of methionine, isoleucine and threonine are not reduced in thedmr1 mutants and the parental line, Ler eds1-2 (Table 1).

Example 3 Pathogen Resistance is Achieved by Application of L-Homoserine

To test if the effect is specific for homoserine the stereo-isomerD-homoserine was tested. Whole seedlings were infiltrated with water, 5mM D-homoserine and 5 mM L-homoserine. Only treatment with the naturalamino acid L-homoserine resulted in resistance to H. parasitica.Seedlings treated with water or D-homoserine did not show a largereduction in pathogen growth and were susceptible to H. parasitica. Theinfiltration was applied to two Arabidopsis accessions, Ler eds1-2 andWs eds1-1, susceptible to Cala2 and Waco9, respectively. Conidiophoreformation was determined as an indicator for H. parasiticasusceptibility. Conidiophores were counted 5 days post inoculation withH. parasitica and 2 days post infiltration with water, D-homoserine orL-homoserine. (FIG. 6). L-homoserine infiltration clearly results inreduction of conidiophore formation and H. parasitica resistance. Thiswas further confirmed by studying pathogen growth in planta by trypanblue staining of Arabidopsis seedlings. Plants were inoculated withisolate Cala2. Two days later the plants were treated by infiltrationwith water, 5 mM D-homoserine, and 5 mM L-homoserine. Symptoms werescored at 5 days post inoculation and clearly showed that only theL-homoserine-infiltrated seedlings showed a strongly reduced pathogengrowth and no conidiophore formation (FIG. 7).

Microscopic analysis showed that only in L-homoserine treated leaves thehaustoria, feeding structures that are made by H. parasitica during theinfection process, are disturbed. Again it is shown that increasedlevels of homoserine in planta lead to pathogen resistance.

Example 4 Identification of HSK Orthologs in Crops 1. Screening ofLibraries on the Basis of Sequence Homology

The nucleotide and amino acid sequences of the homoserine kinase geneand protein of Arabidopsis thaliana are shown in FIGS. 8 and 9 (SEQ IDNOs: 99-100).

Public libraries of nucleotide and amino acid sequences were comparedwith the sequences of FIGS. 8 and 9 (SEQ ID NOs: 99-100).

This comparison resulted in identification of the complete HSK codingsequences and predicted amino acid sequences in Citrus sinensis, Populustrichocarpa (1), Populus trichocarpa (2), Solanum tuberosum (2), Solanumtuberosum (1), Nicotiana benthamiana, Ipomnoea nil, Glycine max,Phaseolus vulgaris, Pinus taeda, Zea mays, and Oryza sativa. Thesequence information of the orthologous proteins thus identified isgiven in FIG. 1. For many other plant species orthologous DNA fragmentscould be identified by BlastX as reciprocal best hits to the Arabidopsisor other plant HSK protein sequences.

2. Identification of Orthologs by Means of Heterologous Hybridisation

The HSK DNA sequence of Arabidopsis thaliana as shown in FIG. 8 (SEQ IDNO: 99) is used as a probe to search for homologous sequences byhybridization to DNA on any plant species using standard molecularbiological methods. Using this method orthologous genes are detected bysouthern hybridization on restriction enzyme-digested DNA or byhybridization to genomic or cDNA libraries. These techniques are wellknown to the person skilled in the art. As an alternative probe the HSKDNA sequence of any other more closely related plant species can be usedas a probe.

3. Identification of Orthologs by Means of PCR

For many crop species, partial HSK mRNA or gene sequences are availablethat are used to design primers to subsequently PCR amplify the completecDNA or genomic sequence. When 5′ and 3′ sequences are available themissing internal sequence is PCR amplified by a HSK specific 5′ forwardprimer and 3′ reverse primer. In cases where only 5′, internal or 3′sequences are available, both forward and reverse primers are designed.In combination with available plasmid polylinker primers, inserts areamplified from genomic and cDNA libraries of the plant species ofinterest. In a similar way, missing 5′ or 3′ sequences are amplified byadvanced PCR techniques, 5′RACE, 3′RACE, TAIL-PCR, RLM-RACE orvectorette PCR.

As an example the sequencing of the Lactuca sativa (lettuce) HSK cDNA isprovided. From the Genbank EST database at NCBI several Lactuca HSK ESTswere identified using the tblastn tool starting with the Arabidopsis HSKamino acid sequence. Clustering and alignment of the ESTs resulted in aconsensus sequence for a 5′HSK fragment and one for a 3′ HSK fragment.To obtain the complete lettuce HSK cDNA the RLM-RACE kit (Ambion) wasused on mRNA from lettuce seedlings. The 5′ mRNA sequence was obtainedby using a primer that was designed in the 3′HSK consensus sequencederived from ESTs (R1S1a: GCCTTCTTCACAGCATCCATTCC—SEQ ID NO: 1) and the5′RACE primers from the kit. The 3′ cDNA sequence was obtained by usingtwo primers designed on the 5′RACE fragment (Let3 RACEOut:CCOTTGCGGTTAATGAGATT—SEQ ID NO: 2, and Let3RACEInn:TCGTGTTGGTGAATCCTGAA—SEQ ID NO: 3) and the 3′RACE primers from the kit.Based on the assembled sequence new primers were designed to amplify thecomplete HSK coding from cDNA to provide the nucleotide sequence andderived protein sequence as presented in FIG. 10 (SEQ ID NOs: 101-102).A similar approach was a used for Solanum lycopersicum (FIG. 14—SEQ IDNOs: 109-110) and Vitis vinifera (FIG. 11—SEQ ID NOs: 103-104).

The complete HSK coding sequences from more than 10 different plantsspecies have been identified from genomic and EST databases. From thealignment of the DNA sequences, conserved regions in the coding sequencewere selected for the design of degenerate oligonucleotide primers (forthe degenerate nucleotides the abbreviations are according to the IUBnucleotide symbols that are standard codes used by all companiessynthesizing oligonucleotides, G=Guanine, A=Adenine, T=Thymine,C=Cytosine, R=A or G, Y=C or T, M=A or C, K=G or T, S=C or G, W=A or T,B=C or G or T, D=G or A or T, H=A or C or T, V=A or C or G, N=A or C orG or T).

The procedure for obtaining internal HSK cDNA sequences of a given plantspecies is as follows:

1. mRNA is isolated using standard methods,

2. cDNA is synthesized using an oligo dT primer and standard methods,

3. using degenerate forward and reverse oligonucleotides a PCR reactionis carried out,

4. PCR fragments are separated by standard agarose gel electrophoresisand fragments of the expected size are isolated from the gel,

5. isolated PCR fragments are cloned in a plasmid vector using standardmethods,

6. plasmids with correct insert sizes, as determined by PCR, areanalyzed by DNA sequencing.

7. Sequence analysis using blastX reveals which fragments contain thecorrect internal HSK sequences,

8. The internal DNA sequence can then be used to design gene- andspecies-specific primers for 5′ and 3′ RACE to obtain the complete HSKcoding sequence by RLM-RACE (as described above).

As an example the sequencing of the Cucumis sativus (cucumber) HSK cDNAis provided. For cucumber two primer combinations were successful inamplifying a stretch of internal coding sequence from cDNA; combination1: primer F1Kom (GAYTTTCYTHGGMTGYGCCGT—SEQ ID NO: 4) and M1RC(GCRGCGATKCCRGCRCAGTT—SEQ ID NO: 5), and combination 2: primer M1Kom(AACTGYGCYGGMATCGCYGC—SEQ ID NO: 6) and R1Kom(CCATDCCVGGAATCAANGGVGC—SEQ ID NO: 7). After cloning and sequencing ofthe amplified fragments cucumber HSK-specific primers were designed for5′ RACE (Cuc5RACEOut: AGAGGATTTTACTAAGTTATTCGTG—SEQ ID NO: 8 andCuc5RACEInn: AGACATAATCTCCCAAGCCATCA—SEQ ID NO: 9) and 3′ RACE(Cuc3RACEOut: TGATGGCTTGGGAGATATGTCT—SEQ ID NO: 10 and Cuc3RACEInn:CACGAATAAACTTAGTAAAAATCCTCT—SEQ ID NO: 11). Finally the completecucumber HSK cDNA sequence was amplified and sequenced (FIG. 12—SEQ IDNOs: 105-106). A similar approach was a used for spinach, Spinaciaoleracea (FIG. 13—SEQ ID NOs: 107-108).

Orthologs identified as described in this example can be modified usingwell-known techniques to induce mutations that reduce the HSK expressionor activity. Alternatively, the genetic information of the orthologs canbe used to design vehicles for gene silencing. All these sequences arethen used to transform the corresponding crop plants to obtain plantsthat are resistant to Oomycota.

Example 5 Reduction of Homoserine Kinase Expression in Arabidopsis bymeans of RNAi

The production of HSK silenced lines has been achieved in Arabidopsis byRNAi. A construct containing two ˜750 bp fragments of the HSK exon inopposite directions was successfully transformed into the ArabidopsisCol-0 accession. The transformants were analysed for resistance to H.parasitica, isolate Waco9. Several transgenic lines were obtained thatconfer resistance to H. parasitica. Analysis of HSK expression andhomoserine accumulation confirm that in the transformed lines the HSKgene is silenced, resulting in resistance to H. parasitica.

Example 6 Mutation of Seeds

Seeds of the plant species of interest are treated with a mutagen inorder to introduce random point mutations in the genome. Mutated plantsare grown to produce seeds and the next generation is screened forincreased accumulation of homoserine. This is achieved by measuringlevels of the amino acid homoserine, by monitoring the level of HSK geneexpression, or by searching for missense mutations in the HSK gene bythe TILLING method, by DNA sequencing, or by any other method toidentify nucleotide changes.

The selected plants are homozygous or are made homozygous by selfing orinter-crossing. The selected homozygous plants with increased homoserinelevels are tested for increased resistance to the pathogen of interestto confirm the increased disease resistance.

Example 7 Transfer of a Mutated Allele into the Background of a DesiredCrop

Introgression of the desired mutant allele into a crop is achieved bycrossing and genotypic screening of the mutant allele. This is astandard procedure in current-day marker assistant breeding of crops.

Tables

TABLE 2 GI numbers (GenInfo identifier) and Genbank accession number forExpressed Sequence Tags (ESTs) and mRNA sequences of the Arabidopsis HSKmRNA and orthologous sequences from other plant species. Species Commonname Detail GI number Genbank Arabidopsis thaliana Thale cress mRNA39104571 AK117871 Citrus sinensis Sweet Orange ESTs 55935768 CV88664228618675 CB293218 55935770 CV886643 28619455 CB293998 Glycine maxSoybean ESTs 10846810 BF069552 17401269 BM178051 8283472 BE02103116348965 BI974560 7285286 AW597773 58024665 CX711406 58017647 CX70438920449357 BQ253481 16105339 BI893079 37996979 CF808568 37996460 CF8080496072786 AW102173 26057235 CA800149 6455775 AW186458 6072724 AW1021119203587 BE329811 Ipomoea nil Japanese moming glory ESTs 74407098CJ761918 74402449 CJ757269 74402115 CJ756935 74388670 CJ743490 NicotianaTobacco ESTs 39880685 CK295868 Benthamiana 39859026 CK284950 39864851CK287885 39864855 CK287887 39859024 CK284949 39864853 CK287886 39880683CK295867 39864849 CK287884 Oryza sativa Rice mRNA 50916171 XM_46855032970537 AK060519 Phaseolus vulgaris Common Bean ESTs 62708660 CV53525662710636 CV537232 62708052 CV534648 62709395 CV535991 62710761 CV53735762708535 CV535131 62708534 CV535130 62711318 CV537914 62707924 CV53452062710733 CV537329 62709601 CV536197 62709064 CV535660 62708834 CV535430Pinus taeda Loblolly Pine ESTs 70780626 DR690274 67490638 DR09226748933532 CO162991 34354980 CF396563 67706241 DR117931 17243465 BM15811534349136 CF390719 66981484 DR057917 48932595 CO162054 66689208 DR01170248933450 CO162909 34350236 CF391819 67706323 DR118013 48932678 CO16213766981399 DR057832 34354850 CF396433 Populus trichocarpa 1 Poplar Genomev1.0, LG_IX, 149339-148242 Expression confirmed by ESTs Populustrichocarpa 2 Poplar Genome v1.0, scaffold_66, 1415935-1417032Expression confirmed by ESTs Solanum tuberosum 1 Potato ESTs 66838966DR037071 61238361 DN588007 39804315 CK251362 39801776 CK250065 9250052BE340521 39832341 CK275363 21917848 BQ116921 9249876 BE340345 39815050CK258070 39804985 CK251702 39804987 CK251703 39825384 CK268406 39832342CK275364 66838967 DR037072 9250394 BE340863 39804317 CK251363 39825385CK268407 21375072 BQ516203 Solanum tuberosum 2 Potato ESTs 39813353CK256373 39793361 CK246131 39793359 CK246130 39813352 CK256372 Zea MaysMaize ESTs 76071237 DT948407 76913306 DV165065 71446162 DR82721271449720 DR830770 78117576 DV535963 91048486 EB158904 71439095 DR82014576936546 DV174774 76012246 DT939416 78085419 DV513812 71766843 DR96478076924795 DV170131 71449067 DR830117 91875652 EB405609 71450175 DR83122578103551 DV521979 78090555 DV518929 78104654 DV523072 76926251 DV17076878111568 DV529965 71773353 DR971257 71425952 DR807002 93282458 EB67472278074199 DV502633 76293328 DV032896 78075462 DV503896 91054750 EB16516886469295 DY235665 74243218 DT651132 74242899 DT650813 101384764 EB81442891054750 EB165168 71440426 DR821476 78121780 DV540164 78103550 DV52197886469794 DY235664 91877777 EB407734 67014441 CO443190 76924794 DV17013076021236 DT948406 71446161 DR827211 78110960 DV529358 78074736 DV50317071428043 DR809093 86469052 DY235422 71440425 DR821475 78121779 DV54016378104653 DV523071 37400920 CF637820 78074198 DV502632 71449719 DR830769Solanum lycopersicum Tomato 58213736 BP877213 7333245 AW621598 4386685AI482761 Unigene SGN-U223239 Sequence described in this patent from SolGenomics Network application Lactuca sativa Lettuce Sequence describedin this patent application Vitis vinifera Grape vine Sequence describedin this patent application Spinacia oleracea Spinach Sequence describedin this patent application Cucumis sativus Cucumber Sequence describedin this patent application A GI number (genInfo identifier, sometimeswritten in lower case, “gi”) is a unique integer which identifies aparticular sequence. The GI number is a series of digits that areassigned consecutively to each sequence record processed by NCBI. The GInumber will thus change every time the sequence changes. The NCBIassigns GI numbers to all sequences processed into Entrez, includingnucleotide sequences from DDBJ/EMBL/GenBank, protein sequences fromSWISS-PROT, PIR and many others. The GI number thus provides a uniquesequence identifier which is independent of the database source thatspecifies an exact sequence. If a sequence in GenBank is modified, evenby a single base pair, a new GI number is assigned to the updatedsequence. The accession number stays the same. The GI number is alwaysstable and retrievable. Thus, the reference to GI numbers in the tableprovides a clear and unambiguous identification of the correspondingsequence.

TABLE 3Primer sequences on insertion/deletion (INDEL, size difference indicated in brackets)markers and cleaved amplified polymorphics sequences (CAP, polymorphic restrictionsite indicated in brackets) used in the mapping of the DMR1 locus.Primer name: BAC Forward SEQ Reverse SEQ TYPE GI number ofand/or TAIR At code primer sequence ID NO: primer sequence ID NO:(size/enzyme) TAIR At code T24112 AATCCAAATTTCTT 12 AAACGAAGAGTGAC 13INDEL 18398180 (At2g16670) GCGAGAACACA 14 AATGGTTGGAG 15 (33) F5J6CCGTCAGATCAGTC 16 CAGAAGCTGATGAT 17 INDEL 23506018 (AT2g17370-80)CTCATCTTGTT 18 CGTGGAAAGTA 19 (30) 30679966 F6P23 CGGTTTCATGTCGA 20AAGAAGAGAACTGC 21 INDEL 22325728 (AT2g17060) GGAAGATCATA 22 GTCAACCTTCC23 (37) T23A1 TCCTTCCATGTCCG 24 AACAAATTTGCTTC 25 INDEL 42570808(AT2g17220-30) AAACCA 26 CAGCCTTT 27 (26) AT2g17190 GAATAGAGGTTGAT 28CTCTTGTATGTTTT 29 CAP 30679898 GGAAATCAAGA 30 ACTGGGCTGAT 31 (MseI)AT2g17200 CCTCTCCACCCATT 32 CGATCCATTTCGTC 33 CAP 30679902 TCTAATTTCG 34AAGCAATCTAC 35 (MboII) AT2g17270 GATGCAGCTAAATT 36 ACGAAAATATCAAA 37 CAP30679927 ATCAGTGTGAA 38 AAGCTCCTTC 39 (NlaIII) AT2g17300-05AGGTAGGATGGTAT 40 GCATGTTTTCTCTA 41 CAP 30679937 TATGTTTGAACT 42AGCGATAGAAG 43 (EcoRI) 22325732 AT2g17310 ATGGGTAACGAAAG 44CACATGTATAAGGT 45 CAP 42569097 AGAGGATTAGT 46 CTTCCCATAGA 47 (MseI)AT2g17360 CCAACAAGTATCCT 48 CCACATCAAACTTA 49 CAP 30679959 CTTTTGTTGTT50 ATGAACTCCAC 51 (MaeIII)

TABLE 4Primer sequences used for amplifying and sequencing of eight candidate DMR1genes for which the TAIR and GI codes are indicated Primer namePrimer sequence SEQ ID NO: TAIR codes GI codes MvD17230_FTTCCCGAAGTGTACATTAAAAGCTC 52 At2g17230 30679913 MvD17230_RTATGTCATCCCCAAGAGAAGAAGAC 53 At2g17230 30679913 MvD17240_FCAATAAAAGCCTTTAAAAGCCCACT 54 At2g17240 30679916 MvD17240_RTAGCTTCTGAAACTGTGGCATTACA 55 At2g17240 30679916 MvD17250_1FCATGATTTGAGGGGTATATCCAAAA 56 At2g17250 22325730 MvD17250_1RGGAGGTGGGATTTGAGATAAAACTT 57 At2g17250 22325730 MvD17250_2FTAGCCTAGAACTCTCTGTTCGCAAG 58 At2g17250 22325730 MvD17250_2RCATTATTTTGCGTAGTTGTGAGTGG 59 At2g17250 22325730 MvD17250_3FCGAAGAAATCCTACAATCAACCATC 60 At2g17250 22325730 MvD17250_3RTCTCACAATTCCCATCTCTTACTCC 61 At2g17250 22325730 MvD17260_1FTTACTCATTTGGGTGAACAGAACAA 62 At2g17260 30679922 MvD17260_1RATCATCCCTAATCTCTCTGCTTCCT 63 At2g17260 30679922 MvD17260_2FGATTAAGATACGGGGAATGGAGTCT 64 At2g17260 30679922 MvD17260_2RATGCAGACAAATAAGATGGCTCTTG 65 At2g17260 30679922 MvD17260_3FGTTGTTGCTCCTGTCACAAGACTTA 66 At2g17260 30679922 MvD17260_3RGAACAAAGACGAAGCCTTTAAACAA 67 At2g17260 30679922 MvD17265_FGAGGACTGCATCTAGAAGACCCATA 68 At2g17265 18398362 MvD17265_RTGGGCTCTCAACTATAAAGTTTGCT 69 At2g17265 18398362 MvD17270_F1TAACGGTAAAGCAACGAATCTATCC 70 At2g17270 30679927 MvD17270_R1TCAAACTGATAACGAGAGACGTTGA 71 At2g17270 30679927 MvD17270_F2TTGCGTTCGTTTTTGAGTCTTTTAT 72 At2g17270 30679927 MvD17270_R2AAACCAGACTCATTCCTTTGACATC 73 At2g17270 30679927 MvD17280_F1TTTAGGATCTCTGCCTTTTCTCAAC 74 At2g17280 42569096 MvD17280_R1GAGAAATCAATAGCGGGAAAGAGAG 75 At2g17280 42569096 MvD17280_F2GCTTAAATAGTCCTCCTTTCCTTGC 76 At2g17280 42569096 MvD17280_R2TCTGCTGGTTCTCATGTTGATAGAG 77 At2g17280 42569096 MvD17290_F1CTCTCCTTCATCATTTCACAAATCC 78 At2g17290 30679934 MvD17290_R1TTCCTCTCGCTGTAATGACCTCTAT 79 At2g17290 30679934 MvD17290_F2TGCCACAGGTGTTGACTATGC 80 At2g17290 30679934 MvD17290_R2TGCTCTTAAACCCGCAATCTC 81 At2g17290 30679934 MvD17290_F3GAAGATGGCTTTAAAGGTCAGTTTGT 82 At2g17290 30679934 MvD17290_R3AGCAACAACAACTAAAAGGTGGAAG 83 At2g17290 30679934

1. An isolated plant which is resistant to a pathogen, wherein the plant has an increased endogenous L-homoserine level as compared to a plant that is not resistant to said pathogen, wherein said plant is selected from the group consisting of cucumber, grape, and tomato, and wherein when said plant is cucumber, said pathogen is Pseudoperonospora cubensis and said cucumber plant has a mutation in the homoserine kinase gene of SEQ ID NO: 105 lowering the homoserine kinase activity of SEQ ID NO: 106; wherein when said plant is grape, said pathogen is Plasmopara viticola and said grape plant has a mutation in the homoserine kinase gene of SEQ ID NO: 103 lowering the homoserine kinase activity of SEQ ID NO: 104; and wherein when said plant is tomato, said pathogen is Phytophthora infestans and said tomato plant has a mutation in the homoserine kinase gene of SEQ ID NO: 109 lowering the homoserine kinase activity of SEQ ID NO:
 110. 2. The plant of claim 2, wherein the mutation in the homoserine kinase gene leads to an ammo acid substitution in the encoded protein.
 3. A method for obtaining a plant which is resistant to a pathogen, wherein the plant has an increased endogenous L-homoserine level as compared to a plant that is not resistant to said pathogen, wherein said plant is selected from the group consisting of cucumber, grape, and tomato, the method comprising: increasing the endogenous L-homoserine level in a cucumber plant by a mutation in the homoserine kinase gene of SEQ ID NO: 105 lowering the homoserine kinase activity of SEQ ID NO: 106 or reducing the expression of SEQ ID NO: 105 to produce a cucumber plant which is resistant to Pseudoperonospora cubensis; or increasing the endogenous L-homoserine level in a grape plant by a mutation in the homoserine kinase gene of SEQ ID NO: 103 lowering the homoserine kinase activity of SEQ ID NO: 104 or reducing the expression of SEQ ID NO: 103 to produce a grape plant which is resistant to Plasmopara viticola; or increasing the endogenous L-homoserine level in a tomato plant by a mutation in the homoserine kinase gene of SEQ ID NO: 109 lowering the homoserine kinase activity of SEQ ID NO: 110 or reducing the expression of SEQ ID NO: 109 to produce a tomato plant which is resistant to Phytophthora infestans.
 4. The method of claim 3, wherein the mutation results in one or more amino acid changes that lead to a lower homoserine kinase activity.
 5. The method of claim 3, wherein the mutation is effected by mutagenic treatment of the cucumber plant, grape plant, or tomato plant.
 6. The method of claim 5, wherein the mutagenic treatment is effected with a mutagen or radiation. 